Recent data has demonstrated the existance of a regulatory T cell subset that protects against various autoimmune diseases including type I diabetes. Very little is known about this population of T cells, however. At present they can only be partially purified using the general T cell markers CD4, CD25, and CD62L. If we could purify these cells to homogeneity, we could begin to study their properties and determine how to use them for therapeutic intervention. This would be especially useful for children identified as being at risk for type I diabetes. Here we propose 2 novel strategies to obtain information about this elusive cell population. (1) SELEX (Systematic Evolution of Ligands by EXponential enrichment) is performed by incubating a target protein with a large pool of degenerate DNA oligomers which fold into 3 dimensional shapes (aptamers) which can bind to ligand with high affinity and specificity. An iterative binding and PCR amplification step is performed to generate a family of aptamers which fold into shapes that bind to the target with high affinity. By combining proteomics with SELEX, we can quickly assess aptamers which bind to useful targets and use them to select subpopulations of CD4+CD25+ cells. We will then assess the ability of this subpopulation to protect NODscid mice from developing diabetes through introduction of pathogenic NOD splenocytes. If we are successful, we will then identify the aptamer target by Mass Spectrometry. (2) We propose to identify critical signal transduction pathways required for regulatory function. We have developed a strain of NOD mouse in which primary T cells can be infected with adenoviral constructs ex vivo with high efficiency. We will use this system to introduce dominant negative mutants to block specific signaling pathways in the CD4+CD25+ population and assess regulatory function in our NODscid assay. We will test three specific hypotheses. (a) regulatory function requires signaling through the TCR, (b) regulatory function requires new gene transcription, and (c) regulatory function requires cell division. We have dominant negative mutants to test each of these hypotheses. If either of these two strategies is successful, we will generate important information regarding the nature of CD4+CD25+ regulatory cells. This information will bring us closer to understanding the biology of these cells and to determining their therapeutic potential.